- Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain
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Administration of the current tuberculosis (TB) vaccine to newborns is not a reliable route for preventing TB in adults. The conversion of XMP to GMP is catalyzed by guaA-encoded GMP synthetase (GMPS), and deletions in the Shiguella flexneri guaBA operon led to an attenuated auxotrophic strain. Here we present the cloning, expression, and purification of recombinant guaA-encoded GMPS from Mycobacterium tuberculosis (MtGMPS). Mass spectrometry data, oligomeric state determination, steady-state kinetics, isothermal titration calorimetry (ITC), and multiple sequence alignment are also presented. The homodimeric MtGMPS catalyzes the conversion of XMP, MgATP, and glutamine into GMP, ADP, PP i, and glutamate. XMP, NH4+, and Mg2+ displayed positive homotropic cooperativity, whereas ATP and glutamine displayed hyperbolic saturation curves. The activity of ATP pyrophosphatase domain is independent of glutamine amidotransferase domain, whereas the latter cannot catalyze hydrolysis of glutamine to NH3 and glutamate in the absence of substrates. ITC data suggest random order of binding of substrates, and PPi is the last product released. Sequence comparison analysis showed conservation of both Cys-His-Glu catalytic triad of N-terminal Class I amidotransferase and of amino acid residues of the P-loop of the N-type ATP pyrophosphatase family.
- Franco, Tathyana Mar A.,Rostirolla, Diana C.,Ducati, Rodrigo G.,Lorenzini, Daniel M.,Basso, Luiz A.,Santos, Diogenes S.
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- Histidine and thermal copolymers of amino acids containing histidine as prebiotic inhibitor for the template-directed formation of oligoguanylate on a poly(C) template
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Possibility of the cooperative chemical evolution of nucleic acids and proteins has been investigated using the template-directed formation of oligoguanylate with amino acids and thermal copolymers of amino acids, in which strong inhibition by histidine containing thermal copolymer and histidine itself was observed. The inhibition is regarded as prebiotic enzymatic activities for the hydrolysis of activated nucleotide monomer and the formation of pyrophospho-capped oligoguanylate.
- Kawamura, Kunio,Kuranoue, Kazuhiro
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- Characterization of complexes of nucleoside-5′-phosphorothioate analogues with zinc ions
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On the basis of the high affinity of Zn2+ to sulfur and imidazole, we targeted nucleotides such as GDP-β-S, ADP-β-S, and AP3(β-S)A, as potential biocompatible Zn2+-chelators. The thiophosphate moiety enhanced the stability of the Zn2+- nucleotide complex by about 0.7 log units. ATP-α,β-CH 2-γ-S formed the most stable Zn2+-complex studied here, log K 6.50, being ~0.8 and ~1.1 log units more stable than ATP-γ-S-Zn2+ and ATP-Zn2+ complexes, and was the major species, 84%, under physiological pH. Guanine nucleotides Zn2+ complexes were more stable by 0.3-0.4 log units than the corresponding adenine nucleotide complexes. Likewise, AP3(β-S)A-zinc complex was ~0.5 log units more stable than AP3A complex. 1H- and 31P NMR monitored Zn2+ titration showed that Zn 2+ coordinates with the purine nucleotide N7-nitrogen atom, the terminal phosphate, and the adjacent phosphate. In conclusion, replacement of a terminal phosphate by a thiophosphate group resulted in decrease of the acidity of the phosphate moiety by approximately one log unit, and increase of stability of Zn2+-complexes of the latter analogues by up to 0.7 log units. A terminal phosphorothioate contributed more to the stability of nucleotide-Zn2+ complexes than a bridging phosphorothioate.
- Sayer, Alon Haim,Itzhakov, Yehudit,Stern, Noa,Nadel, Yael,Fischer, Bilha
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- High Level Expression of XMP Aminase in Escherichia coli and Its Application for the Industrial Production of 5′-Guanylic Acid
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To improve the efficiency of the enzymatic conversion of 5′-xanthylic acid (XMP) to 5′-guanylic acid (GMP), we attempted to increase the activity of the conversion enzyme, XMP aminase (GMP synthetase) encoded by the guaA gene in Escherichia coli. By connecting the PL promoter of λ phage, the SD sequence of trpL of E. coli, and ATG, at a suitable position upstream of the guaA gene, we obtained plasmid pPLA66. Sequencing of the nucleotides of the upstream region of the guaA gene on pPLA66 showed that the C-terminal region of the guaB gene, which encodes IMP dehydrogenase, was conserved and a short peptide consisted of 14 amino acids was coded. E. coli MP347/pPLA66 showed an increase in the activity of approximately 370 times when compared with that of the strain MM294, and the amount of the enzyme protein represented approx. 34% of the total cellular protein. Strain MP347/pPLA66 was cultivated in a 5-liter jar fermentor using a medium which contained mainly corn steep liquor. The culture broth had high XMP aminase activity. In the conversion reaction using mixed broths consisted of 600ml of XMP-fermentation broth of Corynebacterium ammoniagenes KY13203 and 30 ml of cultured broth of E. coli MP347/pPLA66, a surfactant, Nymeen S-215 and xylene were added to the reaction mixture to make the cell membrane permeable to nucleotides. After 23 h of the reaction, 70mg/ml (131 mM) of GMP·Na2·7H2O was accumulated from 83 mg/ml (155 mM) of XMP·Na3 ·7H2O, without addition of ATP. The molar conversion yield was approx. 85%. The facts that the cell membrane was treated to allow nucleotides to permeate and that the conversion reaction proceeded well enough in spite of a small amount of E. coli cells indicate ATP was regenerated from AMP by C. ammoniagenes cells and supplied to E. coli cells. Therefore, it was considered that the coupling reaction between these two kind of strains was established.
- Fujio, Tatsuro,Nishi, Tatsunari,Ito, Seiga,Maruyama, Akihiko
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- Catalytic activity of human guanylate-binding protein 1 coupled to the release of structural restraints imposed by the C-terminal domain
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Human guanylate-binding protein 1 (hGBP-1) shows a dimer-induced acceleration of the GTPase activity yielding GDP as well as GMP. While the head-to-head dimerization of the large GTPase (LG) domain is well understood, the role of the rest of the protein, particularly of the GTPase effector domain (GED), in dimerization and GTP hydrolysis is still obscure. In this study, with truncations and point mutations on hGBP-1 and by means of biochemical and biophysical methods, we demonstrate that the intramolecular communication between the LG domain and the GED (LG:GED) is crucial for protein dimerization and dimer-stimulated GTP hydrolysis. In the course of GTP binding and γ-phosphate cleavage, conformational changes within hGBP-1 are controlled by a chain of amino acids ranging from the region near the nucleotide-binding pocket to the distant LG:GED interface and lead to the release of the GED from the LG domain. This opening of the structure allows the protein to form GED:GED contacts within the dimer, in addition to the established LG:LG interface. After releasing the cleaved γ-phosphate, the dimer either dissociates yielding GDP as the final product or it stays dimeric to further cleave the β-phosphate yielding GMP. The second phosphate cleavage step, that is, the formation of GMP, is even more strongly coupled to structural changes and thus more sensitive to structural restraints imposed by the GED. Altogether, we depict a comprehensive mechanism of GTP hydrolysis catalyzed by hGBP-1, which provides a detailed molecular understanding of the enzymatic activity connected to large structural rearrangements of the protein. Database: Structural data are available in RCSB Protein Data Bank under the accession numbers: 1F5N, 1DG3, 2B92.
- Ince, Semra,Zhang, Ping,Kutsch, Miriam,Krenczyk, Oktavian,Shydlovskyi, Sergii,Herrmann, Christian
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p. 582 - 599
(2020/06/02)
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- Helices on Interdomain Interface Couple Catalysis in the ATPPase Domain with Allostery in Plasmodium falciparum GMP Synthetase
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GMP synthetase catalyses the conversion of XMP to GMP through a series of reactions that include hydrolysis of Gln to generate ammonia in the glutamine amidotransferase (GATase) domain, activation of XMP to adenyl-XMP intermediate in the ATP pyrophosphatase (ATPPase) domain and reaction of ammonia with the intermediate to generate GMP. The functioning of GMP synthetases entails bidirectional domain crosstalk, which leads to allosteric activation of the GATase domain, synchronization of catalytic events and tunnelling of ammonia. Herein, we have taken recourse to the analysis of structures of GMP synthetases, site-directed mutagenesis and steady-state and transient kinetics on the Plasmodium falciparum enzyme to decipher the molecular basis of catalysis in the ATPPase domain and domain crosstalk. Our results suggest an arrangement at the interdomain interface, of helices with residues that play roles in ATPPase catalysis as well as domain crosstalk enabling the coupling of ATPPase catalysis with GATase activation. Overall, the study enhances our understanding of GMP synthetases, which are drug targets in many infectious pathogens.
- Shivakumaraswamy, Santosh,Pandey, Nivedita,Ballut, Lionel,Violot, Sébastien,Aghajari, Nushin,Balaram, Hemalatha
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p. 2805 - 2817
(2020/06/25)
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- Synthesis of ribonucleotides from the corresponding ribonucleosides under plausible prebiotic conditions within self-assembled supramolecular structures
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Abiotic synthesis of ribonucleotides, mainly at the 5′ position, from the corresponding ribonucleosides within self-assembled supramolecular structures, based on guanosine:borate hydrogels, was carried out in the temperature range of 70-90 °C, using urea and a phosphate source (K2HPO4 or hydroxyapatite). Phosphorylation is possible at initial concentrations of guanosine lower than 20 mM and it is more efficient using wet/dry cycles. Monoamidophosphate (and, eventually, diamidophosphate), diamidodiphosphate and pyrophosphate are intermediates in the synthesis of ribonucleotides. These conclusions are supported by NMR spectroscopy and mass spectrometry analysis of samples. On the other hand, after reaction, hydrogels can produce globular aggregates by the addition of water and decreasing temperature, thus confirming that ribonucleotides, once activated under suitable conditions, could form polyribonucleotides.
- Franco,Ascenso,Ilharco,Da Silva
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supporting information
p. 2206 - 2209
(2020/02/20)
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- Thermophilic phosphoribosyltransferases Thermus thermophilus HB27 in nucleotide synthesis
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Phosphoribosyltransferases are the tools that allow the synthesis of nucleotide analogues using multi-enzymatic cascades. The recombinant adenine phosphoribosyltransferase (TthAPRT) and hypoxanthine phosphoribosyltransferase (TthHPRT) from Thermus thermophilus HB27 were expressed in E.coli strains and purified by chromatographic methods with yields of 10-13 mg per liter of culture. The activity dependence of TthAPRT and TthHPRT on different factors was investigated along with the substrate specificity towards different heterocyclic bases. The kinetic parameters for TthHPRT with natural substrates were determined. Two nucleotides were synthesized: 9-(β-D-ribofuranosyl)-2-chloroadenine 5'-monophosphate (2-l-AMP) using TthAPRT and 1-(β-Dribofuranosyl)pyrazolo[3,4-d]pyrimidine-4-one 5'-monophosphate (Allop-MP) using TthPRT.
- Fateev, Ilja V.,Sinitsina, Ekaterina V.,Bikanasova, Aiguzel U.,Kostromina, Maria A.,Tuzova, Elena S.,Esipova, Larisa V.,Muravyova, Tatiana I.,Kayushin, Alexei L.,Konstantinova, Irina D.,Esipov, Roman S.
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p. 3098 - 3105
(2019/01/21)
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- Cloning, expression and biochemical characterization of xanthine and adenine phosphoribosyltransferases from Thermus thermophilus HB8
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Purine phosphoribosyltransferases, purine PRTs, are essential enzymes in the purine salvage pathway of living organisms. They are involved in the formation of C-N glycosidic bonds in purine nucleosides-5′-monophosphate (NMPs) through the transfer of the 5-phosphoribosyl group from 5-phospho-α-D-ribosyl-1-pyrophosphate (PRPP) to purine nucleobases in the presence of Mg2+. Herein, we report a simple and thermostable process for the one-pot, one-step synthesis of some purine NMPs using xanthine phosphoribosyltransferase, XPRT or adenine phosphoribosyltransferase, APRT2, from Thermus thermophilus HB8. In this sense, the cloning, expression and purification of TtXPRT and TtAPRT2 is described for the first time. Both genes, xprt and aprt2 were expressed as his-tagged enzymes in E. coli BL21(DE3) and purified by a heat-shock treatment, followed by Ni-affinity chromatography and a final, polishing gel-filtration chromatography. Biochemical characterization revealed TtXPRT as a tetramer and TtAPRT2 as a dimer. In addition, both enzymes displayed a strong temperature dependence (relative activity >75% in a temperature range from 70 to 90 °C), but they also showed very different behaviour under the influence of pH. While TtXPRT is active in a pH range from 5 to 7, TtAPRT2 has a high dependence of alkaline conditions, showing highest activity values in a pH range from 8 to 10. Finally, substrate specificity studies were performed in order to explore their potential as industrial biocatalyst for NMPs synthesis.
- Del Arco, Jon,Martinez, María,Donday, Manuel,Clemente-Suarez, Vicente Javier,Fernández-Lucas, Jesús
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p. 216 - 223
(2017/09/30)
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- A unique choanoflagellate enzyme rhodopsin exhibits lightdependent cyclic nucleotide phosphodiesterase activity
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Photoactivated adenylyl cyclase (PAC) and guanylyl cyclase rhodopsin increase the concentrations of intracellular cyclic nucleotides upon illumination, serving as promising secondgeneration tools in optogenetics. To broaden the arsenal of such tools, it is desirable to have light-activatable enzymes that can decrease cyclic nucleotide concentrations in cells. Here, we report on an unusual microbial rhodopsin that may be able to meet the demand. It is found in the choanoflagellate Salpingoeca rosetta and contains a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. We examined the enzymatic activity of the protein (named Rh-PDE) both in HEK293 membranes and whole cells. Although Rh-PDE was constitutively active in the dark, illumination increased its hydrolytic activity 1.4-fold toward cGMP and 1.6-fold toward cAMP, as measured in isolated crude membranes. Purified full-length Rh-PDE displayed maximal light absorption at 492 nm and formed the M intermediate with the deprotonated Schiff base upon illumination. The M state decayed to the parent spectral state in 7 s, producing long-lasting activation of the enzyme domain with increased activity. We discuss a possible mechanism of the Rh-PDE activation by light. Furthermore, Rh-PDE decreased cAMP concentration in HEK293 cells in a light-dependent manner and could do so repeatedly without losing activity. Thus, Rh-PDE may hold promise as a potential optogenetic tool for light control of intracellular cyclic nucleotides (e.g. to study cyclic nucleotide-associated signal transduction cascades).
- Yoshida, Kazuho,Tsunoda, Satoshi P.,Brown, Leonid S.,Kandori, Hideki
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p. 7531 - 7541
(2017/05/12)
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- Fluorescent Sensing of Guanine and Guanosine Monophosphate with Conjugated Receptors Incorporating Aniline and Naphthyridine Moieties
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Ethyne-linked naphthyridine-aniline conjugated molecules are selective sensors of decylguanine in dichloromethane and guanosine monophosphate in water (Kass = 16 000 M-1). The 2-acetamido-1,8-naphthyridine moiety binds with guanine in a DAA-ADD triply hydrogen-bonded motif. The aniline moiety enhances an electron-donating effect, and the substituent is tuned to attain extra hydrogen bonds, π-π stacking, and electrostatic interactions. The proposed binding modes are supported by a Job plot, ESI-MS, 1H NMR, UV-vis, and fluorescence spectral analyses.
- Lu, Shao-Hung,Phang, Riping,Fang, Jim-Min
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supporting information
p. 1724 - 1727
(2016/05/19)
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- Experimental and Computational Evidence for a Loose Transition State in Phosphoroimidazolide Hydrolysis
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Phosphoroimidazolides play a critical role in several enzymatic phosphoryl transfer reactions and have been studied extensively as activated monomers for nonenzymatic nucleic acid replication, but the detailed mechanisms of these phosphoryl transfer reactions remain elusive. Some aspects of the mechanism can be deduced by studying the hydrolysis reaction, a simpler system that is amenable to a thorough mechanistic treatment. Here we characterize the transition state of phosphoroimidazolide hydrolysis by kinetic isotope effect (KIE) and linear free energy relationship (LFER) measurements, and theoretical calculations. The KIE and LFER observations are best explained by calculated loose transition structures with extensive scissile bond cleavage. These three-dimensional models of the transition state provide the basis for future mechanistic investigations of phosphoroimidazolide reactions.
- Li, Li,Lelyveld, Victor S.,Prywes, Noam,Szostak, Jack W.
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supporting information
p. 3986 - 3989
(2016/05/19)
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- Mode of action of recombinant hypoxanthine-guanine phosphoribosyltransferase from Mycobacterium tuberculosis
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Tuberculosis (TB) is the second most important cause of mortality worldwide due to a single infectious agent, Mycobacterium tuberculosis. A better understanding of the purine salvage pathway can unveil details of the biology of M. tuberculosis that might be used to develop new strategies to combat this pathogen. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is an enzyme from the purine phosphoribosyltransferase (PRTase) family and catalyzes the conversion of hypoxanthine or guanine and 5-phospho-α-d-ribose 1-diphosphate (PRPP) to, respectively, inosine 5′-monophosphate (IMP) or guanosine 5′-monophosphate (GMP), and pyrophosphate (PPi). Gel filtration chromatography has shown that recombinant M. tuberculosis HGPRT (MtHGPRT) is homodimeric. A sequential compulsory ordered enzyme mechanism with PRPP as the substrate that binds to free MtHGPRT enzyme and PPi as the first product to dissociate is proposed based on kinetic data and thermodynamics of ligand binding from isothermal titration calorimetry (ITC) results. ITC data have also provided thermodynamic signatures of non-covalent interactions for PRPP, IMP and GMP binding to free MtHGPRT. Thermodynamic activation parameters (Ea, ΔG#, ΔS#, ΔH#) for the MtHGPRT-catalyzed chemical reaction, pre-steady-state kinetics, solvent kinetic isotope effects, equilibrium constants and pH-rate profiles are also presented. Pre-steady-state analysis reveals that there is an initial rapid phase (burst) followed by a slower phase, suggesting that product release is rate limiting. The data here described provide a better understanding of the mode of action of MtHGPRT.
- Patta, Paulo C.,Martinelli, Leonardo K. B.,Rotta, Mariane,Abbadi, Bruno L.,Santos, Diogenes S.,Basso, Luiz A.
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p. 74671 - 74683
(2015/09/21)
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- Fully automated continuous meso-flow synthesis of 5′-nucleotides and deoxynucleotides
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The first continuous meso-flow synthesis of natural and non-natural 5′-nucleotides and deoxynucleotides is described, representing a significant advance over the corresponding in-flask method. By means of this meso-flow technique, a synthesis with time consumption and high-energy consumption becomes facile to generate products with great efficiency. An abbreviated duration, satisfactory output, and mild reaction conditions are expected to be realized under the present procedure.
- Zhu, Chenjie,Tang, Chenglun,Cao, Zhi,He, Wei,Chen, Yong,Chen, Xiaochun,Guo, Kai,Ying, Hanjie
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p. 1575 - 1581
(2015/02/19)
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- The reaction of activated RNA species with aqueous fluoride ion: A convenient synthesis of nucleotide 5′-phosphorofluoridates and a note on the mechanism
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The chemistry of 5′-phosphorimidazolides of ribonucleosides is extended to include their reaction with alkali metal fluorides in aqueous solution. High yields of 5′-phosphorofluoridates are formed, especially with potassium fluoride, but no detectable oligomerization products were formed. A combination of HPLC, mass spectrometry, synthesis, kinetics, and NMR confirms the identities of the products. Judicious control of pH leads to higher yields in shorter reaction times. This new methodology contrasts favorably with other synthetic routes involving non-aqueous chemistry or aqueous chemistry with a nucleotide triphosphate.
- Aldersley, Michael F.,Joshi, Prakash C.,Schwartz, Herbert M.,Kirby, Anthony J.
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p. 1464 - 1466
(2014/03/21)
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- An RNA modification with remarkable resistance to RNase A
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A 3′-deoxy-3′-C-methylenephosphonate modified diribonucleotide is highly resistant to degradation by spleen phosphodiesterase and not cleaved at all by snake venom phosphodiesterase. The most remarkable finding is that, despite the fact that both the vicinal 2-hydroxy nucleophile and the 5′-oxyanion leaving group are intact, the 3′-methylenephosponate RNA modification is also highly resistant towards the action of RNase A.
- Ghidini, Alice,Ander, Charlotte,Winqvist, Anna,Stroemberg, Roger
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supporting information
p. 9036 - 9038
(2013/09/24)
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- Synthesis of oligoribonucleotides with phosphonate-modified linkages
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Solid phase synthesis of phosphonate-modified oligoribonucleotides using 2′-O-benzoyloxymethoxymethyl protected monomers is presented in both 3′→5′ and 5′→3′ directions. Hybridisation properties and enzymatic stability of oligoribonucleotides modified by regioisomeric 3′- and 5′-phosphonate linkages are evaluated. The introduction of the 5′-phosphonate units resulted in moderate destabilisation of the RNA/RNA duplexes (ΔTm -1.8 °C/mod.), whereas the introduction of the 3′-phosphonate units resulted in considerable destabilisation of the duplexes (ΔTm -5.7 °C/mod.). Molecular dynamics simulations have been used to explain this behaviour. Both types of phosphonate linkages exhibited remarkable resistance in the presence of ribonuclease A, phosphodiesterase I and phosphodiesterase II.
- Pav, Ondej,Koiova, Ivana,Barvik, Ivan,Pohl, Radek,Budinsky, Milo,Rosenberg, Ivan
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supporting information; experimental part
p. 6120 - 6126
(2011/10/10)
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- RETRACTED ARTICLE: First prebiotic generation of a ribonucleotide from adenine, d-ribose and trimetaphosphate
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Adenosine monophosphate isomers are obtained by self-assembling of adenine, d-ribose and trimetaphosphate in aqueous solution in good yields. This generation of a ribonucleotide from its three molecular components occurs in a one-pot reaction at room temperature for about 30-40 days and with high chemio-, regio-, and stereo-selectivity. Similar results are obtained with guanine. A mechanism is also proposed.
- Baccolini, Graziano,Boga, Carla,Micheletti, Gabriele
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supporting information; experimental part
p. 3640 - 3642
(2011/05/04)
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- Biosynthetic origin and mechanism of formation of the aminoribosyl moiety of peptidyl nucleoside antibiotics
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Several peptidyl nucleoside antibiotics that inhibit bacterial translocase I involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. We present here the delineation of the biosynthetic pathway for this moiety upon in vitro characterization of four enzymes (LipM-P) that are functionally assigned as (i) LipO, an l-methionine:uridine-5′-aldehyde aminotransferase; (ii) LipP, a 5′-amino-5′-deoxyuridine phosphorylase; (iii) LipM, a UTP:5-amino-5-deoxy-α-d-ribose-1-phosphate uridylyltransferase; and (iv) LipN, a 5-amino-5-deoxyribosyltransferase. The cumulative results reveal a unique ribosylation pathway that is highlighted by, among other features, uridine-5′-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor.
- Chi, Xiuling,Pahari, Pallab,Nonaka, Koichi,Van Lanen, Steven G.
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supporting information; experimental part
p. 14452 - 14459
(2011/11/04)
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- A reversed phase hplc method for the analysis of nucleotides to determine 5'-PDE enzyme activity
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5'-Phosphodiesterase (5'-PDE) can be extracted from barley roots and used as a catalyst in the hydrolysis of RNA to produce 5'-nucleotides. The assay of enzyme activity is essential for the production of 5'PDE. To improve the conventional assays, we developed and validated a new method for the analysis of 5'-PDE enzyme activity using reversed phased high performance liquid chromatography (RP-HPLC). The method is based on the quantification of the four 5'-nucleotides namely cytidine 5'-monophosphate (5'-CMP), uridine 5'monophosphate (5'-UMP), guanosine 5'-mono-phosphate (5'-GMP) and adenosine 5'-mono-phosphate (5'AMP), produced in the enzymatic hydrolysis of yeast RNA. The optimal condition for the enzymatic hydrolysis of RNA to detect the enzyme activity was investigated. The results show that when the hydrolysis takes place at 70 °C for 30 min at pH 5.0, the hydrolysis reaction has highest yield for the four of the 5'-nucleotides. 5'-PDE demonstrated highest catalytic capability. These four 5'-nucleotides were utilized for the analysis of enzyme activity of 5'-PDE with our newly developed HPLC method. Excellent reproducibility, precision, and linearity were obtained for this HPLC method.
- Hua, Jie,Huang, Ke-Long
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experimental part
p. 167 - 174
(2011/11/29)
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- An improved one-pot synthesis of nucleoside 5'-triphosphate analogues
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Nucleoside 5'-triphosphate (NTP) analogues are valuable tools for biochemical and medicinal research. Therefore, a facile and efficient synthesis of NTP analogues is required. Here, we report on an improved nucleoside 5'-triphosphorylation procedure to obtain pure products after liquid chromotagrpahy (LC) separation with no need for high performance liquid chromatography (HPLC) purification. To improve the selectivity of the reaction we attempted the optimization of several parameters such as solvent, pyrophosphate nucleophilicity, time and temperature of the reaction. Eventually, the reaction was optimized by decreasing the temperature to -15°C and increasing the reaction time to 2 hours, based on monitoring time-dependent product distribution using 31P NMR. Furthermore, the NTPs were obtained as pure products after LC separation, which was impossible in the original Ludwig procedure. Good yields were obtained for all studied natural and synthetic nucleosides.
- Gillerman, Irina,Fischer, Bilha
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p. 245 - 256
(2011/08/06)
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- Crystal Structure of the ATPPase Subunit and Its Substrate-Dependent Association with the GATase Subunit: A Novel Regulatory Mechanism for a Two-Subunit-Type GMP Synthetase from Pyrococcus horikoshii OT3
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Guanosine 5′-monophosphate synthetase(s) (GMPS) catalyzes the final step of the de novo synthetic pathway of purine nucleotides. GMPS consists of two functional units that are present as domains or subunits: glutamine amidotransferase (GATase) and ATP pyrophosphatase (ATPPase). GATase hydrolyzes glutamine to yield glutamate and ammonia, while ATPPase utilizes ammonia to convert adenyl xanthosine 5′-monophosphate (adenyl-XMP) into guanosine 5′-monophosphate. Here we report the crystal structure of PH-ATPPase (the ATPPase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon Pyrococcus horikoshii OT3). PH-ATPPase consists of two domains (N-domain and C-domain) and exists as a homodimer in the crystal and in solution. The N-domain contains an ATP-binding platform called P-loop, whereas the C-domain contains the xanthosine 5'-monophosphate (XMP)-binding site and also contributes to homodimerization. We have also demonstrated that PH-GATase (the glutamine amidotransferase subunit of the two-subunit-type GMPS from the hyperthermophilic archaeon P. horikoshii OT3) alone is inactive, and that all substrates of PH-ATPPase except for ammonia (Mg2+, ATP and XMP) are required to stabilize the active complex of PH-ATPPase and PH-GATase subunits.
- Maruoka, Shintaro,Horita, Shoichiro,Lee, Woo Cheol,Nagata, Koji,Tanokura, Masaru
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experimental part
p. 417 - 429
(2011/01/03)
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- What is the conformation of physiologically-active dinucleoside polyphosphates in solution? Conformational analysis of free dinucleoside polyphosphates by NMR and molecular dynamics simulations
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Dinucleoside polyphosphates, or dinucleotides (NpnN′; N, N′ = A, U, G, C; n = 2-7), are naturally occurring ubiquitous physiologically active compounds. Despite the interest in dinucleotides, and the relevance of their conformation to their biological function, the conformation of dinucleotides has been insufficiently studied. Therefore, here we performed conformational analysis of a series of NpnN′ Na+ salts (N = A, G, U, C; N′ = A, G, U, C; n = 2-5) by various NMR techniques. All studied dinucleotides, except for Up4/5U, formed intramolecular base stacking interactions in aqueous solutions as indicated by NMR. The conformation around the glycosidic angle in NpnN′s was found to be anti/high anti and the preferred conformation around the C4′-C5′, C5′-O5′ bonds was found to be gauche-gauche (gg). The ribose moiety in NpnN′s showed a small preference for the S conformation, but when attached to cytosine the ribose ring preferred the N conformation. However, no predominant conformation was observed for the ribose moiety in any of the dinucleotides. Molecular dynamics simulations of Ap2A and Ap4A Na+ salts supported the experimental results. In addition, three modes of base-stacking were found for Ap2/4A: α-α, β-β and α-β, which exist in equilibrium, while none is dominant. We conclude that natural, free NpnN′s (n = 2-5) at physiological pH exist mostly in a folded (stacked), rather than extended conformation, in several interconverting stacking modes. Intramolecular base stacking of NpnN′s does not alter the conformation of each of the nucleotide moieties, which remains the same as that of the mononucleotides in solution.
- Stern, Noa,Major, Dan Thomas,Gottlieb, Hugo Emilio,Weizman, Daniel,Fischer, Bilha
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experimental part
p. 4637 - 4652
(2010/12/19)
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- Natural occurrence of 2′,5′-linked heteronucleotides in marine sponges
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2′,5′-oligoadenylate synthetases (OAS) as a component of mammalian interferon-induced antiviral enzymatic system catalyze the oligomerization of cellular ATP into 2′,5′-linked oligoadenylates (2-5A). Though vertebrate OASs have been characterized as 2′-nucleotidyl transferases under in vitro conditions, the natural occurrence of 2′,5′-oligonucleotides other than 2-5A has never been demonstrated. Here we have demonstrated that OASs from the marine sponges Thenea muricata and Chondrilla nucula are able to catalyze in vivo synthesis of 2-5A as well as the synthesis of a series 2′,5′-linked heteronucleotides which accompanied high levels of 2′,5′-diadenylates. In dephosphorylated perchloric acid extracts of the sponges, these heteronucleotides were identified as A2′p5′G, A2′p5′U, A2′p5′C, G2′p5′A and G2′p5′U. The natural occurrence of 2′-adenylated NAD+ was also detected. In vitro assays demonstrated that besides ATP, GTP was a good substrate for the sponge OAS, especially for OAS from C. nucula. Pyrimidine nucleotides UTP and CTP were also used as substrates for oligomerization, giving 2′,5′-linked homo-oligomers. These data refer to the substrate specificity of sponge OASs that is remarkably different from that of vertebrate OASs. Further studies of OASs from sponges may help to elucidate evolutionary and functional aspects of OASs as proteins of the nucleotidyltransferase family.
- Lopp, Annika,Reintamm, Tonu,Kuusksalu, Anne,Tammiste, Indrek,Pihlak, Arno,Kelve, Merike
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experimental part
p. 235 - 254
(2010/10/19)
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- Selective synthesis of phosphate monoesters by dehydrative condensation of phosphoric acid and alcohols promoted by nucleophilic bases
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(Chemical Equation Presented) Phosphate monoesters are synthesized from a mixture of phosphoric acid (1 or 2 equiv) and alcohols (1 equiv) in the presence of tributylamine. The reaction is promoted by nucleophilic bases such as N-alkylimidazole and 4-(N,N-dialkylamino)pyridine. 2′,3′-I- Isopropylidene ribonucleosides are selectively converted to their 5′-monophosphates without the protection of amino groups in nucleobases.
- Sakakura, Akira,Katsukawa, Mikimoto,Ishihara, Kazuaki
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p. 1999 - 2002
(2007/10/03)
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- Borate-nucleotide complex formation depends on charge and phosphorylation state
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Flow injection analysis with electrospray ionization mass spectrometry was used to investigate borate-nucleotide complex formation. Solutions containing 100 μM nucleotide and 500 μM boric acid in water-acetonitrile-triethylamine (50:50:0.2, v/v/v; pH 10.3
- Kim, Danny H.,Faull, Kym F.,Norris, Andrew J.,Eckhert, Curtis D.
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p. 743 - 751
(2007/10/03)
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- Macrocyclic amines as catalysts of the hydrolysis of the triphosphate bridge of the mRNA 5′-cap structure
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The reactions of a 5′-cap model compound P 1-(7-methylguanosine) P3-guanosine 5′,5′-triphosphate, m7GpppG, were studied in the presence of three different macrocyclic amines (2-4) under neutral conditions. The only products observed in the absence of the macrocycles resulted from the base-catalysed imidazole ring-opening and the acid-catalysed cleavage of the N7-methylguanosine base, whereas in the presence of these catalysts hydrolysis of the triphosphate bridge predominated. The latter reaction yielded guanosine 5′-monophosphate, guanosine 5′-diphosphate, 7-methylguanosine 5′-monophosphate and 7-methylguanosine 5′-diphosphate as the initial products, indicating that both of the phosphoric anhydride bonds were cleaved. The overall catalytic activity of all three macrocycles was comparable. The hydrolysis to guanosine 5′-diphosphate and 7-methylguanosine 5′-monophosphate was slightly more favoured than the cleavage to yield guanosine 5′-monophosphate and 7-methylguanosine diphosphate. All the macrocycles also enhanced the subsequent hydrolysis of the nucleoside diphosphates, 2 being more efficient than 3 and 4.
- Zhang, Zhibo,Loennberg, Harri,Mikkola, Satu
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p. 3404 - 3409
(2007/10/03)
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- Kinetic analysis of the temperature dependence of the template-directed formation of oligoguanylate from the 5′-phosphorimidazolide of guanosine on a poly(C) template with Zn2+
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A kinetic study of the temperature dependence of the template-directed formation of oligoguanylate (oligo(G)) on polycytidylic acid (poly(C)) from the 5′-phosphorimidazolide of guanosine (ImpG) has been carried out in the presence of Zn2+ at 40
- Kawamura,Umehara
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p. 927 - 935
(2007/10/03)
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- Phosphorylation of guanosine using guanosine-inosine kinase from Exiguobacterium acetylicum coupled with ATP regeneration.
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Guanosine 5'-monophosphate (5'-GMP) and inosine 5'-monophosphate (5'-IMP) are widely used as flavor enhancers. Recently, a novel process for 5'-IMP production by phosphorylation of inosine using guanosine-inosine kinase coupled with ATP regeneration was reported. In this study, we demonstrated the practical possibility of producing 5'-GMP by phosphorylation of guanosine using a guanosine-inosine kinase from Exiguobacterium acetylicum coupled with ATP regeneration.
- Kawasaki,Usuda,Shimaoka,Utagawa
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p. 2259 - 2261
(2007/10/03)
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- Interactions between aminocalixarenes and nucleotides or nucleic acids
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Four calixarenes with (trimethylammonium)methyl groups at the phenyl rings in the upper rim were prepared. Association constants K with mononucleotides were determined in D2O by NMR shift titration, partially also by fluorescence competition titration using ANS as dye. The complexation free energies ΔG obtained with the derivatives of the calix[4]cone (AC4c) and the calix[4]-1,3-alternate (AC4a) conformation were similar, but increased from AMP (18 ± 1 kJ mol-1) to ADP (20 ± 1 kJ mol-1), to ATP (22 ± 1 kJ mol-1]. With the calix[6] derivative (AC6) the corresponding values were 22, 24, 27 kJ mol-1, with the calix[8] host (AC8) 24, 26, 28 kJ mol-1, respectively. The large contribution of salt bridging to the complexation was obvious from the ΔG difference between adenosine and e.g. AMP (with the calix[4]cone derivative 5.6 and 17.7 kJ mol-1, respectively). Affinity differences between different nucleobases increased moderately with the size of the macrocyclic host, e.g. ΔΔG between AMP and TMP was 1 kJ mor-1 with calix[4]cone, 2 kJ mor-1 with calix[6], and 3 kJ mol-1 with calix[8] compounds. The results are in line with computer simulated complex structures in which the nucleobase or sugar parts are only partially inserted into the calix cavity. This agrees with the observed complexation induced NMR shifts (CIS), which are small but increase with the ring size of the host. Noticeably the CIS values are substantially larger for much weaker bound nucleosides. Affinities of the four aminocalixarenes with double-stranded calf thymus (CT) DNA, with polydA*polydT and with polydG*polydC were characterized by ΔTm of the double-strand denaturation temperature and by fluorimetric assays using ethidium bromide (C50 values). The calix[4]cone derivative AC4c shows, due to the four positive charges converging at one side, the strongest effects. They surpass spermine although this also bears four protonated ammonium groups, indicating additional binding contributions from the phenyl moieties. The larger, more flexible calix[6]- and calix[8]-derivatives AC6 and AC8 show only small affinity increases in spite of their 6 or 8 positive charges. Preliminary molecular modeling studies indicate that based on the distances between the ammonium centers only partial contact of all centers to the groove phosphates can materialize. The ligands AC4c, AC4a and AC6 exhibit a remarkable preference for DNA in comparison to RNA mimics.
- Shi, Youheng,Schneider, Hans-Joerg
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p. 1797 - 1803
(2007/10/03)
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- Unique Catalysis and Regioselectivity Observed in the Poly(C)-Directed RNA Dimer Formation from 2-MeImpG: Kinetic Analysis as a Function of Monomer and Polymer Concentration
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Polycytidylate, poly(C), serves as a scaffold or template to direct and catalyze the synthesis of long oligoguanylates from guanosine 5′-phosphate 2-methylimidazolide, 2-MeImpG. In the absence of poly(C), small amounts of three isomeric dimers, i.e., the 2′-5′-, the 3′-5′-, and the pyrophosphate-linked, are formed slowly. In the presence of poly(C) oligomers that are primarily 3′-5′-linked are formed quickly and in high yield. Product analysis suggests that the oligomers are elongation products of the 3′-5′-linked dimer, abbreviated D. Assuming that D is formed slowly from two molecules of 2-MeImpG (Scheme 1) and elongates relatively fast, the initial rate of dimerization, d[D]/dt in M h-1, was determined using two independent methods. The first method is based on the approximation that at the onset of the reaction the substrate is consumed only via hydrolysis and dimerization, and thus elongation can be neglected. The second, more accurate, method exploits the assertion that every oligomer was once a 3′-5′-linked dimer. Hence the concentration of D was obtained indirectly from the concentration of the oligomer products. These two methods gave comparable results. Experiments were run in aqueous solution in the presence of 1.0 M NaCl, 0.2 M MgCl2 at pH 7.9 ± 0.1 and 23°C. Controls were run in the absence of poly(C) and in the presence of other polynucleotides. The kinetics were determined as a function of both monomer and polymer concentration the latter expressed in C equivalents. The kinetic data obtained in the presence of poly(C) confirmed an earlier conclusion regarding the remarkable effect of poly(C) on the formation of the 3′-5′-linked diguanylate. Initial dimerization rates were quantitatively correlated using a simple template-directed (TD) model that presumes cooperative binding (two association constants) of 2-MeImpG on poly(C) and reaction between adjacent template-bound molecules. The model allows for the estimation of the association constants and the intrinsic rate constant of dimerization, k2*- Insights into the detailed mechanism are also gained from this analysis. The fact that the proposed model can successfully correlate kinetic data that vary by more than 5000-fold between the slowest and the fastest reaction adds confidence and suggests the suitability of this model for describing TD reactions in general. It is anticipated that similar analysis of other known TD reactions may lead to clues that will facilitate the design of more efficient polynucleotide-synthesizing systems.
- Kanavarioti, Anastassia,Baird, Eldon E.,Hurley, T. Brian,Carruthers, Julie A.,Gangopadhyay, Sumana
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p. 8323 - 8333
(2007/10/03)
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- The Cu2+-promoted cleavage of mRNA 5'-cap analogs: A kinetic study with p1(7-methylguanosin-5'-yl) p3-(nucleosid-5'-yl) triphospates and p1- (7-methylguanosin-5'-yl) p4-(guanosin-5'-yl) tetraphosphate
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A kinetic study on the cleavage of a number of mRNA 5'-cap analogs, m7GpppN and m7GppppG, with Cu2+ aquo ion has been performed. Time- dependent product distributions at various pH and metal ion concentrations have been determined by capillary zone electrophoresis, and these data have been used to calculate the rate constants for various parallel reactions of the breakdown of the cap analogs.
- Wieczorek, Zbigniew,Darzynkiewicz, Edward,Kuusela, Satu,Loennberg, Harri
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- Hydrolytic Susceptibilities of Modified 5'-mRNA Cap Analogues to the Yellow Lupin Ap4A Hydrolases
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Several new dinucleoside tri- and tetraphosphates, 5'-mRNA cap analogues, have been tested as potential substrates for two enzymes: highly specific homogeneous Ap3A and (asymmetrical) Ap4A hydrolase from yellow lupin seeds.The Ap3A hydrolase cleaves all examined dinucleotide triphosphates yielding in most cases GMP or m7GMP and modified diphosphates as the main hydrolysis products (75-100percent).Only m7Gp3G has been hydrolyzed randomly.Compounds with unmodified guanosine have been degraded at comparable rate as natural substrate Ap3A.For the analogues modified in guanosine or sugar moiety significant differences have been observed in the rate of hydrolysis.Ap4A hydrolase seems to be more specific enzyme than Ap3A hydrolase.Dinucleoside tetraphosphates containing guanosine or 7-methylguanosine have been degraded more slowly than Ap4A.In contrast, di- and trimethylated compounds have been hydrolyzed with the rate 2-fold higher in comparison with Ap4A.For all methylated dinucleoside tetraphosphates, GTP and methylated nucleoside monophosphates have been found as the main hydrolysis products.
- Bojarska, Elzbieta,Stepinski, Janusz,Guranowski, Andrzej,Starzynska, Elzbieta,Chlebicka, Lidia,et al.
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p. S192 - S196
(2007/10/03)
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- Regiodefined synthesis and conformational properties of adenyldiyl trimers with unsymmetrical 2'-5' and 3'-5' internucleotide linkages
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Adenyldiyl trimers with different kinds of substituents on 2'-5' and 3'-5' phosphate linkages have been synthesized in a general, regiodefined manner. Examination of the 2D NMR spectra reveals that the trimers with adenyl(2'-5')adenosine linkage make a syn-anti as well as syn-syn base stack between the two adenyl bases and exist as a mixture of the two conformers.
- Hayakawa,Hirose,Noyori
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p. 9899 - 9916
(2007/10/02)
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- Structure-activity relationships for the binding of ligands to xanthine or guanine phosphoribosyl-transferase from Toxoplasma gondii
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Preliminary characterization of Toxoplasma gondii phosphoribosyltransferase activity towards purine nucleobases indicates that there are at least two enzymes present in these parasites. One enzyme uses hypoxanthine, guanine, and xanthine as substrates, while a second enzyme uses only adenine. Furthermore, competition experiments using the four possible substrates suggest that there may be a third enzyme that uses xanthine. Therefore, sixty-eight purine analogues and thirteen related derivatives were evaluated as ligands of T. gondii phosphoribosyltransferase, using xanthine or guanine as substrates, by examining their ability to inhibit these reactions in vitro. Inhibition was quantified by determining apparent K(i) values for compounds that inhibited these activities by greater than 10% at a concentration of 0.9 mM. On the basis of these data, a structure-activity relationship for the binding of ligands to these enzymes was formulated using hypoxanthine (6-oxopurine) as a reference compound. It was concluded that the following structural features of purine analogues are required or strongly preferred for binding to both enzymes: (1) a pyrrole-type nitrogen (lactam form) at the 1-position; (2) a methine (=CH-), a pyridine type nitrogen (=N-), or an exocyclic amino or oxo group at the 2-position; (3) no exocyclic substituents at the 3-position; (4) an exocyclic oxo or thio group in the one or thione tautomeric form at the 6-position; (5) a pyridine-type nitrogen (=N-) or a methine group at the 7-position; (6) a methine group at the 8-position; (7) a pyrrole-type nitrogen or a carbon at the 9-position; and (8) no exocyclic substituents at the 9-position. These findings provide the basis for the rational design of additional ligands of hypoxanthine, guanine, and xanthine phosphoribosyltransferase activities in T. gondii.
- Naguib,Iltzsch,El Kouni,Panzica,El Kouni
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p. 1685 - 1693
(2007/10/03)
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- Phosphorylation of nucleosides with phosphorus oxychloride in trialkyl phosphate
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The reaction of guanosine and triethyl phosphate at 50°C for 15 min produced the guanosine-triethyl phosphate complex in which triethyl phosphate is coordinated to guanosine of high-anti form in a 1:1 molar ratio. During the conversion of guanosine to guanosine 5'-monophosphate with phosphorus oxychloride, the guanosine-triethyl phosphate complex showed excellent selectivity and high reactivity toward phosphorus oxychloride compared with those of guanosine. The rate of selective phosphorylation of guanosine into guanosine 5'-monophosphate was markedly improved by preheating the mixture of guanosine and triethyl phosphate at 50°C, followed by adding phosphorus oxychloride to the mixture at 0°C. Thus, the 5'-phosphorylation of guanosine with phosphorus oxychloride in triethyl phosphate is considered to progress via the guanosine-triethyl phosphate complex as the reaction intermediate.
- Ikemoto,Haze,Hatano,Kitamoto,Ishida,Nara
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p. 210 - 215
(2007/10/02)
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- Catalysis of Hydrolysis and Nucleophilic Substitution at the P-N Bond of Phosphoimidazolide-Activated Nucleotides in Phosphate Buffers
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Phosphoimidazolide-activated derivatives of guanosine and cytidine 5'-monophosphates, henceforth called ImpN's, exhibit enhanced rates of degradation in the presence of aqueous inorganic phosphate in the range 4.0 (*) pH (*) 8.6.This degradation is been attributed to (i) nucleophilic substitution of the imidazolide and (ii) catalysis of the P-N bond hydrolysis by phophate.The first reaction results in the formation of nucleoside 5'-diphosphate and the second in nucleoside 5'-monophosphate.Analysis of the observed rates as well as the product ratios as a function of pH and phosphate concentration allow distinction between various mechanistic possibilities.The results show that both H2PO4(-) and HPO4(2-) participate in both hydrolysis and nucleophilic substitution.Statistically corrected bimolecular rate constants indicate that the dianion is 4 times more effective as a general base than the monoanion, and 8 times more effective as nucleophile.The low Bronsted value β = 0.15 calculated for these phosphate species, presumed to act as general bases in facilitating water attack, is consistent with the fact that catalysis of the hydrolysis of the P-N bond in ImpN's has not been detected before.The βnuc = 0.35 calculated for water, H2PO4(-), HPO4(2-), and hydroxide acting as nucleophiles indicates a more associative transition state for nucleotidyl (O2POR(-) with R = nucleoside) transfers than that observed for phosphoryl (PO3(2-)) transfers (βnuc = 0.25).With respect to the stability/reactivity of ImpN's under prebiotic conditions, our study shows that these materials would not suffer additional degradation due to inorganic phophate, assuming the concentrations of phosphate, Pj, on prebiotic Earth were similar to those in the present oceans (j> ca. 2.25 μM).
- Kanavarioti, Anastassia,Rosenbach, Morgan T.
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p. 1513 - 1521
(2007/10/02)
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- Phosphorylation of Nucleotides with Inorganic Cyclo-Triphosphate
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Phosphorylation of nucleotides (nucleoside 3'- and 5'-monophosphates, and 2'-deoxynucleoside 5'-monophosphates) with inorganic sodium cyclotriphosphate (P3m) was studied in aqueous solutions under various conditions (mixing ratio of P3m to nucleotides, pH, reaction temperature, and time). (1) Unprotected nucleoside 5'-monophosphates (5'-NMP's) were easily phosphorylated at the cis-2',3'-diol by P3m to form selectively nucleoside 2',5'-bis(monophosphate) (2',5'-NDP's), nucleoside 3',5'-bis(monophosphate) (3',5'-NDP's), and nucleoside 2',3'-cyclic 5'-bis(monophosphate) (cNDP's). (2) The phosphorylation of 5'-NMP's was strongly dependent on mixing ratio, pH, reaction temperature, and time.Under conditions of high mixing ratios of P3m to 5'-NMP's (5:1 - 10:1), high pH (12), and room temperature, 92 -98percent of 5'-NMp's was converted into 3',5'-NDP's and 2',5'-NDP's in roughly equimolar quantities. (3) Small quantities (5 -8percent) of cNdP's were formed at the initial stage of reaction of 5'-NMP's with P3m but in the course of the reaction for a long period, cNDP's were hydrolyzed to 2',5'-NDP's and 3',5'-NDP's. (4) Nucleoside 3'-monophosphates (3'-NMP's) and 2'-deoxynucleoside 5'-monophosphates (dNMP's) could not be phosphorylated by P3m, which indicates that the presence of hydroxyl groups at both 2'- and 3'-positions on nucleotides is indispensable for the phosphorylation of nucleotides with P3m. (5) The mechanism of the formation of 2',5'-NDP's, 3',5'-NDP's, and cNDP's in the phosphorylation of 5'-NMP's with P3m is discussed.
- Tsuhako, Mitsutomo,Kunitomi, Rumi,Baba, Yoshinobu,Miyajima, Tohru
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p. 490 - 497
(2007/10/02)
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- IMMUNOAFFINITY PURIFICATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE FROM LACTUCA COTYLEDONS
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To facilitate further study of a multifunctional phosphodiesterase, previously partially purified from Lactuca cotyledons, a new purification step has been devised.This uses an immunoaffinity column based upon polyclonal antibodies raised against the partially purified enzyme.Preparation of the immunoaffinity column, purufication of the enzyme using the new protocol, and analysis of the activity of the purified enzyme are described.The additional step produced an enzyme preparation with a significantly higher specific activity and free of nucleotidase and non-specific phosphatase activity.The observed properties of the enzyme confirm similarities with mammalian multifunctional phosphodiesterase but reaffirm the existence of two types of substrate binding site on the Lactuca phosphodiesterase.Key Word Index - Lactuca sativa; Compositae; lettuce; cotyledons; cyclic nucleotides; phosphodiesterase; immunoaffinity purification; 3',5'-cyclic AMP; 3',5'-cyclic GMP; 3',5'-cyclic CMP; 3',5'-cyclic UMP.
- Chiatante, Donato,Balconi, Carlotta,Newton, Russell P.,Brown, Eric G.
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p. 2477 - 2484
(2007/10/02)
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- A GENERAL APPROACH TO NUCLEOSIDE 3'- AND 5'-MONOPHOSPHATES
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Diallyloxyphosphorylation of nucleoside hydroxyls followed by palladium(0)-catalyzed deallylation provides a new, general method for the preparation of the 3'- and 5'-monophosphates.
- Hayakawa, Y.,Wakabayashi, S.,Nobori, T.,Noyori, R.
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p. 2259 - 2262
(2007/10/02)
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- PHOSPHORYLATING AGENT FOR THE SYNTHESIS OF OLIGONUCLEOTIDE WITH ALIPHATIC AMINO GROUP AT 5' END
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A lipophilic phosphorylating agent was prepared and used for the synthesis of pentadeoxyribonucleotide with aminoethyl group at 5' end on a polymer support by the phosphotriester method.
- Tanaka, Toshiki,Tamatsukuri, Shigeru,Ikehara, Morio
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p. 2611 - 2614
(2007/10/02)
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- Studies on Chemical Synthesis of mRNAs. I. Synthesis and Properties of N2-Tritylguanosine Derivatives and Application to Synthesis of pGpUpU
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Trityl (Tr), 4-methoxytrityl (MMTr), and 4,4'-dimethoxytrityl (DMTr) groups were introduced into the 2-amino group of 2',3',5'-tri-O-acetylguanosine by treatment with the corresponding trityl chlorides in pyridine to afford the N2-tritylated guanosine derivatives in high yields.Similarly, the N6-tritylated adenosine derivatives were synthesized.The stability of the three kinds of trityl groups under acidic conditions were described.The MMTr and Tr groups were found to be suitable for the protection of the 2-amino group of guanosine in oligonucleotide synthesis.An appropriately protected N2-tritylguanosine 5-phosphorodithioate derivative was synthesized and utilized for the synthesis of pGpUpU.
- Hata, Tsujiaki,Gokita, Noboru,Sakairi, Nobuo,Yamaguchi, Kazuo,Sekine, Mitsuo,Ishido, Yoshiharu
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p. 2949 - 2955
(2007/10/02)
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- Bis(2,2,2-trichloroethyl) Phosphorochloridite as a Reagent for the Phosphorylation of Oligonucleotides: Preparation of 5'-Phosphorylated 2',5'-Oligoadenylates
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Bis(2,2,2-trichloroethyl) phosphorochloridite was found to be a useful reagent for the phosphorylation of protected nucleosides and oligonucleotides especially when the phosphate blocking group was 2,2,2-trichloroethyl and the hydroxyl protecting group was tert-butyldimethylsilyl.Thus compounds 2a-c,f,g could be phosphorylated to the corresponding 5'-phosphotriesters 4a-c,f,g in yields of 75-99percent.Removal of the protecting groups (to give the 5'-monophosphates 6a-c,f,g) was achieved by zinc-copper couple/2,4-pentanedione/DMF treatment to remove the 2,2,2-trichloroethyl group and tetrabutylammonium fluoride/THF treatment to remove the tert-butyldimethylsilyl groups.A method was found that permits preparation of reproducibly active zinc-copper couple.As a hydroxyl protecting group, the isopropylidene moiety was somewhat less useful in conjunction with the use of bis(2,2,2-trichloroethyl) phosphorochloridite.Thus, upon phosphorylation of 2d and 2e, the phosphotriesters 4d and 4e were obtained in yields of 83percent and 61percent, respectively.Deblocking of the isopropylidene groups was accomplished with formic acid at room temperature to give 6a and 6b in yields of 74percent and 70percent, respectively.The 5'-phosphorylated 2'-5'-linked oligonucleotides 6f and 6g were converted to the corresponding 5'-triphosphates to give compounds 1a and 1b which are found in extracts of interferon-treated cells upon incubation with double-stranded RNA.
- Imai, Jiro,Torrence, Paul F.
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p. 4015 - 4021
(2007/10/02)
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